TARGETED MUTAGENESIS IN ZEBRAFISH BY ANIMAL CLONING

描述（申请人提供）：斑马鱼是研究脊椎动物发育和模拟人类疾病的极好遗传系统。几次大规模的诱变筛选已经产生了数千种在重要生物过程中具有缺陷的突变鱼。然而，该系统仍然缺乏进行反向遗传学和基因标记诱变的有效方法。最近，我们已经表明，正常的斑马鱼个体可以克隆核移植使用供体细胞核从长期培养的细胞。在本提案中，我们将探讨这一发现在两个领域的效用。首先，我们将建立一个基因陷阱程序，通过该程序，培养细胞将被选择用于基因陷阱事件，然后用于通过动物克隆生产斑马鱼。第二，我们将开发斑马鱼反向遗传学的靶向突变方法。通过在培养的成纤维细胞中进行同源重组来破坏许多选定的候选基因，然后将携带同源重组事件的细胞核用于产生克隆的斑马鱼。在过去的三年里，我们的实验室投入了大量的资源和时间来开发斑马鱼的基因靶向技术。一批具有独特技术才能的科学家聚集在一起。在持续的支持下，我们相信我们可以实现拟议的目标，斑马鱼将很快提供小鼠研究社区所享有的所有必要工具。这两个系统将真正相互补充，以促进我们对脊椎动物发育生物学的理解。

DESCRIPTION (provided by applicant): The zebrafish is a superb genetic system for studying vertebrate development and modeling human diseases. Several large-scale mutagenesis screens have generated thousands of mutant fish with defects in important biological processes. However, this system still lacks efficient ways to perform reverse genetics and gene tagged mutagenesis. Recently, we have shown that normal zebrafish individuals can be cloned by nuclear transfer using donor nuclei from long-term cultured cells. In this proposal, we will explore the utility of this finding in two areas. First, we will establish a gene trap procedure by which cultured cells will be selected for gene trap events and then used to produce zebrafish by animal cloning. Second, we will develop targeted mutagenesis methods for reverse genetics in zebrafish. A number of selected candidate genes will be disrupted by homologous recombination in cultured fibroblast cells and the nuclei carrying the homologous recombination events will then be used to produce cloned zebrafish. For the past three years, our laboratory has devoted significant resources and time towards developing gene-targeting technology in zebrafish. A group of scientists with unique technological talents have been assembled. With a continuous support, we believe that we can achieve the proposed goals and zebrafish will soon offer all the essential tools that have been enjoyed by the mouse research community. The two systems will then truly complement each other to advance our understanding of vertebrate developmental biology.